Alu elements represent one of the biggest sources of intrinsic instability in the human genome. The human genome contains over 1,000,000 Alu elements, making up about 11% of the human genome. These approximately 300 bp elements contain variable levels of mismatch relative to one another and are dispersed throughout the genome in all orientations. They are also enriched in genes and many genes are densely populated with these elements in the genome. Thus, non-allelic homology between different Alu elements has the ability to confuse many DNA replication and repair processes leading to higher rates of various genetic instabilities and Alu elements represent one of the major sources of intrinsic genomic instability. Almost 0.5% of new human genetic diseases are the result of non-allelic recombination between nearby (1-50 kb) Alu pairs. In some genes this type of instability is one of the major sources of genetic defects. In addition, both inverted Alu pairs and direct repeat Alu pairs seem to contribute an intrinsic instability that leads to a concentration of NHEJ events in the vicinity of Alu elements. Their length, mismatch and high copy number make Alu elements a relatively unique substrate that leads to genetic variation in humans in primates in ways not experienced in most other genomes and which are therefore understudied. We have developed a powerful reporter gene system that allows us to explore the features of Alu elements that contribute to different forms of genetic instability. In this proposal we use adaptations of that reporter gene to understand Alu orientation, spacing and mismatch influences on deletions caused with both I-Sce1 to create targeted double-strand breaks, as well as replication stress. We will then use a series of very deep sequencing studies, and human tumor deep sequencing studies to provide an overall picture of the factors that lead to higher instability around different Alu variants and how Alu-influenced rearrangements compete with other DNA repair processes. These studies will allow us to develop rules that will predict which regions of the human genome are prone to which types of Alu-influenced genetic instability (NAR vs. NHEJ for instance). Furthermore, we adapt our reporter system to a transgenic mouse reporter system that will, for the first time, allow us to determine whether the germline, somatic cells, and tumors are subject to similar instabilities associated with their Alu elements and begin the process of understanding how genetic variants, particularly in DNA repair may alter how Alus contribute to genetic instability. These data are likely to help design better ways to influence Alu-related genetic instability, perhaps by guiding better designed synthetic lethality therapeutics.